The Tet(L) and Tet(K) proteins are important Gram-positive antibiotic (tetracycline, Tc)-resistance proteins that possess three catalytic modes. They catalyze efflux of: (i) a tetracycline-divalent metal complex in exchange for a greater number of protons [Tc-Me 2+/H+ antiport]; (ii) Na+ or K+ in exchange for a greater number of H+ [Na+(K+)/H+ antiport}; and (iii) Tc-Me2+, Na+ or K+ for a greater number of external K+ [Tc-Me2+ (Na+(K+)K+ antiport, i.e. net K+ uptake]. A comprehensive study of structure-function, physiological impact, and regulation pertaining to the multiple functions of these Tet proteins will contribute fundamental mechanistic insights into this class of transporters. In addition, these studies will enhance approaches to rational design of inhibitors and to assessments of selective pressures, other than Tc, that enhance retention of the tet genes. Specific Aim #1 is to develop structure-function information about Tet(L) and Tet(K) through integrated use of biochemical and site-directed mutagenesis approaches. Deployment of a comprehensive array of vesicle and proteoliposome assays of the three catalytic activities will: probe a ping-pong model and hypothesized involvement of occluded transition states in the catalytic cycles; clarify the coupling stoichiometry, and whether K+ ions can completely substitute for H+; extend the analysis of the critical functions of the Motif C region of the transporter in ion-coupling and substrate preference. Selected mutant Tet proteins will also be examined for their effects on competitive fitness of the natural host relative to wild type Tet(L) or Tet(K). Using a combination of molecular, genetic and biochemical approaches, the oligomeric structure of Tet(L) will be examined. Specific Aim #2 is the continuation of the structural biology initiative which is currently focused primarily on further improvement of the quality and diffraction properties of 3D crystals of Tet(L). Crystals of sufficient quality, perhaps stabilized in a single conformation by inhibitors, substrates or conformationally sensitive monoclonal antibodies, will be analyzed by x-ray diffraction. A parallel track is also to be pursued on 2D crystals followed by cryoelectron microscopy. These studies, conducted while the longer-term 3D effort continues, is expected to provide lower resolution structural information of interest. Specific Aim 3 will: clarify the basis for different phenotypes of tet(L) mutants; will further test the novel translational reinitiation model for tet(L) regulation; and will probe additional, Na- and pH related regulation.